Plant growth stimulating composition and method employing mixtures of a gibberellic acid and purine compound



United States Patent Ofilice Patented Jan. 21, ISfii 3,113,753 PLANT GRUW'I'H STIMULA'IDJG CGMPDSlTIGN AND METHQD EWLGYING TURES OF A GBBERELLIC ACE) AND PURINE COMPOUND William Shire, 843 E. 38th St, and Charles G. Skinner, 44% Buval St, both of Austin, Tex. No Drawing. Filed Apr. 14, 1961, Ser. No. 102,950 14 Claims. (Cl. 712.5)

This invention relates to new compositions of matter. More particularly, it relates to synergistic compositions of a gibberellin and substituted purine derivatives with ctivity which stimulate the rate of germination of seeds. The invention also involves methods of preventing and breaking dormancy and of increasing the rate of rooting, flowering, budding and growth of plants.

Specific conditions of light and temperature are known to be required for enicient germination of many kinds of seed. For example, lettuce seed normally require a light and/ or temperature pre-trc atment in conjunction with water uptake to prepare the dormant seed for germination. These stimulatory conditions may not be present in an abnormal growing season; thus the planted seed will not germinate with the resultant failure to yield a harvest. Other types of seed are similarly dificult to germinate unless relatively specific conditions of light, moisture, temperature, etc. are present. Therefore, compositions and methods of increasing germination are highly desirable, particularly in areas where water content of the soil may be low. Several chemicals have been studied with respect to the breaking of this dormancy effect including gibberellins, substituted tln'oand aminopurines, auxins, vitamins, various amino acids and other chemicals.

We have now found that compositions of gibberellin and substituted purines are substantially superior to either of the components alone in inducing the breaking of dormancy of seed even under favorable external conditions. Further, the concentrations necessary to bring about this stimulatory effect using a synergistic mixture of substituted purine and a gibberellin are substantially lower than those required to induce a comparable stimulation by either of the substances alone within a 24 hour period.

The gibberellins have been described in various publications such as Source Book on Gibberellin by F. H. Stodola, Agricultural Research Service, United States Department of Agriculture, May 1958, particularly publications such as Stodola et al., Arch. of Biochem. and Biophysics 54, 240, 1955. These publications described gibberellins under common names such as Gibberellin A of Gibberellin A 1 1 0 Gibberellin A (C H O and Gibberellin A Gibberellin X and gibberellic acid (G i-1 0 and alkali metal salts. The term gibberellins is a generic term and is intended to cover gibberellic acid compounds having similar properties and the empirical formula:

I H2 C lgHzuO 5 H; L H5 in some instances, the synergistic effects of a mixture of a gibberellin and substituted purines may be augmented by the addition of various natural substances. These include, for example, auxins, vitamins and amino acids, particularly with respect to post-germination growth of the plants.

Pro-soaking Early Curled Simpson lettuce seed in a gibberellin solution, for example gibberellic acid at 30 and 100 'y/ml. (micrograms per milliliter) gives 11 and 15% germination, respectively; and pro-soaking of these seed in 1 and /ml. solutions of 6-benzylaminopurine results in 4 and 23% germination, respectively, over a 24 hour period in the absence of light and at 3'0 C. A

combination of gibberellic acid and 6-benzylaminopurine yields a marked synergistic stimulation of rate of lettuce seed germination; for example, a mixture of 1 'y/ml. of the purine derivative and 30 'y/ml. of gibberellic acid gives 71% germination under identical conditions. A similar synergistic effect has been observed using other variety of seed after pro-treatment with these compositions; for example, carpet grass, endive, oak leaf lettuce and the like.

Various substituted 6-thiopurine derivatives have also been found to be synergistic with a gibberellin in stimulating the rate of seed germination, although, in general, they are effective at somewhat higher concentration levels. Pre-treatment of lettuce seed with a 10 'y/rnl. solution of 6-benzylthiopurine and then allowing the seed to germinate in the dark at 30 C. for 24 hours, results in only 4% germination of the seed. A 'y/ml. solution of gibberellic acid under identical conditions gave 15% germination, while a mixture of 10 'y/ml. of 6- benzylthiopurine and 100 'y/ml. of gibberellic acid produces a synergistic response giving 56% germination under the same non-optimal conditions of temperature and absence of light.

The new compositions of the present invention may be applied to the seed in any one of several ways. For example, they may be applied in aqueous solution or suspension, or in water-organic solvent mixtures. The addition of dilute alkali facilitates solution of the amphoteric purine derivative in water. The compounds may also be suspended in a suitable inert carrier such as talc and applied to the dried seed prior to planting and subsequent Watering.

In addition to the above described efieo't of increasing the rate of seed germination these compositions also stimulate the rooting of certain cuttings. Further, young lettuce plants grown from seed pre-treated in appropriate concentrations of these synergistic compositions, have larger leaves and stems as well as forming a more well developed root system. This latter efiect is in contrast to the effect observed afiter soaking the seed in a gibberellin such as gibberellic acid alone, wherein the root system is frequently abnormal.

The substituted purines which have been found to form synergistic compositions with a gibberellin in affecting plant growth include those of the type -alkyuaminoand 6-alkylthiopurines, 6 dialkylaminopurines, 6 aralkylarninoand 6-aralkylthiopurines, 6 arylaminopurines, 6- heterocyclicallrylaminopurines and these substituted purines having in the 2-position such groups as hydrogen, amino and iluoro radicals. Specific examples of such compounds include 6-pentyland -hexylthiopurine; 6- propyl-, 6-n-butyl-, 6-n-pentyl-, 6-n-hexyl and -n-heptyiaminopurine; 6-dipropylaminopurine; 6 phenyl-, 6-benzyl-, 6-phenylethyland 6-phenylpropylaminopurine; 6-hhexyl, 6-benzyl-, 6-phenylethyland G-phenylpropylthiopurine; 6a-naphthyland ect-naphthylrnethylaminopurine; 6-(2-furfuryl)-, 6-(2-thenyl)-, 6 (2-pyridylmethyl)-, 6- (3-pyridylmethyl)-, 6 (4-pyridylmethyl), Z-amino 6- benzyl-, 2-amino-6-phenylethyl-, Z-amino 6 ()\-pyridylmethyl)- and 2-fiuoro-6-benzylaminopurines. The preparation of these compounds have been described in the chemical literature by the present inventors and others.

The amount of both a gibberellin and a substituted purine derivative used in the synergistic compositions for each treatment will depend upon the specific activity of the purine derivative as well as the biological system which is being treated. These values including optimum concentrations, time of pretreatment and other factors should be determined for each system under consideration prior to field use.

In general, the substituted purines exert a substantial synergizing effect on the plant growth regulating action of gibberellin in amounts as low as 0.1 part by weight of the purine to 100 parts of a gibberellin, although somewhat larger proportions of the thiopurine derivatives are desirable. Proportions of the substituted purines up to at least as much as 30 parts by weight of purine to 1 part of gibberellin may be used. The optimum proportions will be found to vary to some extent with the total concentration of the component in the treating compositions.

The present application is a continuation-in-part of our application Serial No. 735,671, filed May 16, 1958, now abandoned.

Example I The seeds were pre-treated for eight hours in the dark at 25 C. in solutions containing about 30 'y/ml. of gibberellic acid and about 1 'y/ml. of the substituted purine derivative shown. The seeds were then separated from the pre-treatment solution in the dark using a blue light and finally allowed to germinate at 30 C. in the dark for 24 hours. One hundred or more seeds were used in each test, and the number of seed germinated were calculated as a percentage of the total.

Lettuce seed of the variety (Early Curled Simpson) were treated with substituted purine derivatives indicated and their rate of germination were compared with seed treated only with water, or only with gibberellic acid or only with the indicated purine derivative and allowed to germinate under identical conditions. The following table shows the results obtained:

Pre-treatment conditions Average percent of seed ger- Gibberellic acid, 'y/ml. Substituted purine, /1111. Ininated Water only 1 G-n-hexylaminopurine, 1 8 30 do 69 0 G-n-hexylthiopurine, 1 1 30 do 21 0 fi-benzyla-minopurine, 1 9 3n o 71 0 G-benzylthiopurine, 1 0 30 (lo 24 O G-furturylaminopurine, 1 do 69 30 None 11 The more active compositions will activate germination of seed in periods as low as ten minutes but treatment for up to eight hours does not lower the germination activity.

Example 2 Under the conditions described in Example 1 except that about 10 v/ml. of the indicated purine derivatives was mmed with about 30 /ml. of gibberellic acid and then allowing the pre-treated lettuce seed (Early Curled Simpson) to germinate under the conditions described in Ex ample 1, the following results were obtained:

Under the conditions described in Example 1 except that about 100 'y/ml. of gibberellic acid is mixed with 4 about 1 'y/ml. of the purine derivatives, or about 7/ ml. of gibberellic acid with about 1% 'y/ml. of the purine derivative We're used, followed by germination under the conditions described in Example 1, gave synergistic stimulations of germination comparable to those described in Examples 1 and 2.

Example 4 The procedure described in Example 1 was used except that the concentration of gibberellic acid used was about 30 'y/mL, and the substituted purine derivatives used were at a concentration of about 10 v/ml. The purine derivatives used included:

-propylarninopurine 6-butylaminopurine 6-pentaylaminopurine 6-phenylpropylaminopurine 6-thenylaminopurine 6wpyridylmethylaminopurine 6 ,B-pyridylmethylaminopurine o-dipropylaminopurine 6-anilinopurine All of these compositions of gibberellic acid and substituted purine derivatives produced a synergistic stimulation of Early Curled Simpson lettuce seed germination under the experimental conditions described in Example 1.

Example 5 Cotton seed were pre-soaked for 3 hours in each of the following solutions:

( 1) Water (2) 3 'y/Inl. of 6-benzylaminopurine (3) 3O 'y/rnl. of gibberellic acid (4) A combination of 2 and 3 above after which, they were allowed to germinate at 25 C. for two days. The germinated seed were then planted in soil and allowed to grow for about one week, after which they were recovered and the indicated measure of growth determined as an average of about 20 plants.

The germination response of seed to the various types of commerically available gibberellins and substituted purine compositions are indicated below using the germinating conditions described in Example 1. Lettuce seed (oak leaf var.) were treated with solutions of 6-benzylaminopurine and/or the four different samples of gibberellin described as:

Code reference Description of sample G Gibberellic acid, 99%.

G Gibberellin A Lot No. 6892055,

(Abbott Laboratories).

G Gibrel (Trademark), potassium salt of gibberellic acid (Merck & Company, Inc.).

G Gibberellin, B grade, Lot No. 102371,

(California Corporation for Biochemical Research) (described as a mixture of gibberellins A and X).

Seed were presoaked in solutions of 6-benzylaminopurine at about 3 'y/ml. and/or the different gibberellins at concentrations of about 30 'y/ml. or 100 /ml. for about hours in the dark at 25 C. The seed were then drained, blotted dry and placed in petri dishes on filter paper wetted with water and allowed to germinate at 3,1 r e-5's 6. emergence of a radicle could be observed. The following summarized the results obtained:

39 C. for 36 hours in the dark. The percentage of 5 Presoaking solution germination was determined by a visual count of the gglgg f seed; a germinated seed was considered as one in which fi-benzylaminopurine, Gibberellin any evidence of emergence of a radicle could be observed. 7/1111 The following summarizes the results obtained:

1 Water blank c0ntrol O 2- a None 35 3 10- None 28 4- None 100 2 Presoakmg solution 5 a 100 88 6 in 100 92 Gibberellin sample Percent germination o-benzylaminopurine, 'y/ml.

Code Concentra- Example 9 reference tion, y/ml.

Using experimental conditions similar to that described Water blank gh g in Example 1, a synengistic germination response was also 30 0 2 found using various concentrations of 2-arnino-6-(substi- 38 g tuted)a-rninopun'nes (described below) and gibberellic 30 6 acid for the presoakin g solutions. These results are g8 g8 summarized as follows: 3 2 0 6 100 75 100 81 Presoaking solutions 100 85 Germina- 100 77 tion,

Gib percent Purine derivative Cone, berellic (24 hours) 7/ 5% Example 7 7/ 1 1 Using the same experimental procedure as described llg fi comm 100 8 in Example 6 and similar samples of gibberellin with the g-amin -ghgnz flan fir igui8 gone r -fl[l'l.1DO- 611 G a O 6 indicated code reference numbers, oak leaf var. lettuce g f g 10 5 8 seed were presoaked in solutions containing 10 /ml. of i y 0- 10 100 84 1 1 100 Z-amrno-G-(B-phenylethybam 10 100 60 -benzyiammopurine and/ or solutions of 30 'y/m or 2 4 d 1 th 1) 10 100 64 'y/rnl. of the gibberelin samples. The results obtained i w 30 n 19 1 2am1no-6-(fi-Dhenylethy1)annno- 30 None 3 are summanzed as O 2-amino-6-(-y-pyridylmethyDamino 30 None 11 Z-amino-(rbenzylamino- 30 100 65 4O 2-amiuofi-(flphenylethyl)amino- 30 100 2-amino-6-(y-pyridylmethyl)amino 30 100 Presoaking solution Gibberellin sample Percent Example 10 germination 45 fi-benz lamino urine V /m], p Code Qoncentra- Using the assay cond1t10ns described 1D. Example 1,

reference the results indicated below demonstrate that 2-fiuor0-6- benzylaminopurine is also synergistic with gibberellic acid blank g 5 in augmenting the rate of seed germination.

3O 85 30 s4 s0 77 30 82 Germina- 100 77 Presoaking solutions tion, 100 77 percent 100 83 (24 hours) Water blank control 5 a The effect on the rate of seed germmatlon of pre-treat- $533 3;gggffgg ggg i fl fi :2 meat with the several gibberellin samples alone at the 4 2i\ijuorfi tl-be r(iizyllignirigpurine, 10 1111. gib- 85 levels indicated gave a negligible response as indicated in 7/ Example 6. On the other hmd, the new compositions o e esent invention show a hi h ercenta e of i i gg g p c We claim: 1. A composition of matter effective in stimulating Example 8 plant growth which comprises gibberellic acid compounds and a synergistically efiective proportion of a 6-substi- U an exPanmemal FY0536m6 l that a tuted purine selected from the group consisting of those scribed in Example 6, a sample of Grbberelhn A obhaw-Hg the formulae: tained from The University of Tokyo, Department of Agricultural Chemistry, Bunkyo-ku, Tokyo, Japan, was R admixed with the concentrations of 6-benzylaminopurme indicated below, and the resulting solutions were used to l N presoak oak leaf var. lettuce seed. The pre-treated seed were then allowed to germinate in the dark at 30 C. for 011 about 60 hours. The percentage of germination was de- L termined by a visual count of the seed; a germinated \N N seed was considered as one in which any evidence of H #33 I N N kN A H in which R is a member of the group consisting of alkyl radicals of 3 to 7 carbon atoms, phenyl, benzyl, phenylethyl, phenylpropyl, thenyl and pyridylmethyl radicals, R is a member of the group consisting of hydrogen and a lower alkyl radical, R is a member of the group consisting of hydrogen, amino and fluoro radicals and R is a member of the group consisting of alkyl radicals of 3 to 7 carbon atoms and the benzyl radical, said gibberellic acid compounds and 6-substituted purines being present in plant growth stimulating quantities.

2. A composition of matter according to claim 1 wherein the gibberellic acid compounds is an alkali metal salt of gibberell-ic acid.

3. A composition of matter in accordance with claim 1 wherein the 6-substituted purine is 6-benzylaminopurine.

4. A composition of matter effective in stimulating plant growth comprising an alkali metal salt of gibberellic acid and a synergistically efiective proportion of 6-benzylaminopurine the said gibberellic acid salt and 6- benzylaminopu-rine being present in sufficient amounts to form an efiectiye plant growth stimulating composition.

5. A composition of matter effective in stimulating plant growth comprising the potassium salt of gibberellic acid and a synergistically efiective proportion of 6-benzylaminopurine, the combined potassium gibberellate and 6-benzylaminopurine being present in amounts suificient to form an effective plant growth stimulating composition.

6. A com-position of matter efiective in stimulating plant growth comprising the potassium salt of gibberellic acid and a synergistically eiiective proportion of 6-hexylaminopurine, the combined potassium gibberellate and 6-hexylaminopunine being present in amounts suflicient to form an effective plant growth stimulating composition.

7. A composition of matter efiective in stimulating plant growth which comprises gibberellic acid and a synergistically effective proportion of 6-b enzylam inopurine, the combined amounts of gibberellic acid and 6- benzylaminopurine being present in plant growth stimulating quantities.

8. A method of stimulating and accelerating the growth and germination of plants and seeds which comprises treating said plants and seeds with a composition of matter comprising gibberellic acid compounds and a synergistically efiective proportion of a 6-substituted purine selected from the group consisting of those having the formulae:

and

and

in which R is a member of the group consisting of alkyl radicals of 3 to 7 carbon atoms, phenyl, benzyl, phenylethyl, phenylpropyl, thenyl and pyridylmethyl radicals, R is a member of the group consisting of hydrogen and lower alkyl radicals, R is a member of the group consisting of hydrogen, amino and fluoro radicals and R is a member of the group consisting of lower alkyl radicals of 3 to 7 carbon atoms and the benzyl radical, the combined amounts of said gibberellic acid compounds and 6-substituted purine being present in plant growth stimulating quantities.

9. A method in accordance with claim 8 wherein the gibberellic acid compounds is an alkali metal salt of gibberellic acid.

10. A method in accordance with claim 8 wherein the 6-substituted purine is 6-benzylaminopurine.

11. A method of stimulating the germination of plant seeds which comprises treating the seeds with a composition of matter comprising an alkali metal salt of gibberellic acid and a synergisticail'ly effective proportion of 6-benzylaminopun'ne, the combined amounts of alkali metal salt of gibberellic acid and 6-benzylarninopurine being present in plant growth stimulating quantities.

12. A method of stimulating the germination of plant seed which comprises treating the seed with a composition of matter comprising the potassium salt of gibberellic acid and a synergistically elfective proportion of 6-benzylaminopturine, the combined amounts of the potassium salt of gibberell-ic acid and 6-benzylaminopurine being present in plant growth stimulating quantity.

13. A method of stimulating the germination of plant seeds which comprises treating the seeds with a composition of matter comprising the potassium salt of gi berellic acid and a synergistically effective proportion of 6-hexylaminopurine, the combined amounts of the potassium salt of gibberellic acid and 6-hexylaminopunine being present in plant growth stimulating quantity.

14. A method of stimulating the germination of plant seeds which comprises treating the seeds with a composition of matter comprising gibberellic acid and a synergistically eifective proportion of 6-benzylaminopurii1e, the combined amounts of gibberellic acid and 6-benzylaminopurine being present in plant growth stimulating quantity.

References Cited in the file of this patent UNITED STATES PATENTS Brian et al July 8, 1959 Shive et a1 Dec. 27, 1960 Shive et a1 July 18, 1961 OTHER REFERENCES 

1. A COMPOSITION OF MATTER EFFECTIVE IN STIMULATING PLANT GROWTH WHICH COMPRISES GIBBERELLIC ACID COMPOUNDS AND A SYNERGISTICALLY EFFECTIVE PROPORTION OF A 6-SUBSTITUTED PURINE SELECTED FROM THE GROUP CONSISTING OF THOSE HAVING THE FORMULAE: 